SUMMARY Discovering mechanism-based treatments for PD, dementia with Lewy Bodies (DLB), and related a-synucleinopathies is an enormous medical need and a central mission of NINDS. Genetic and neuropathological data implicate accumulation of aS as an essential feature of both idiopathic and sporadic PD. aS missense mutations, increased gene dosage, and certain 5? or 3? UTR regulatory alleles can each significantly heighten PD risk. In 2011, we applied non-denaturing methods to identify a hitherto unrecognized form of cellular aS: a-helical tetramers. This finding raised controversy because two decades of in vitro work on recombinant aS indicated it occurred as a ?natively unfolded? monomer, and because the helical tetramers cannot be observed by any biochemical method that breaks open cells, as that rapidly disassembles the native tetramers. Accordingly, our initial R01 application was not funded because the reviewers stated the tetramer hypothesis was ?too controversial? to warrant NIH funding. An appeal to NINDS Council that this did not reflect NIH guidance on seeking scientific novelty led to an SEP review resulting in a different view: the new concept should be pursued, especially by those who initiated it. About a dozen labs have subsequently published evidence for the existence of aS tetramers. During this first grant period, we have analyzed the complex T:M equilibrium in health and PD. We found that all fPD missense mutations significantly lower the T:M ratio, including in human neurons. The Ko lab (Hopkins) then discovered that Gaucher?s GBA mutations lower the aS T:M ratio in human neurons. Given this progress, we now wish to extend this entirely novel hypothesis to answer certain key questions relevant to PD, DLB and AD. Our proposed new experiments will be enabled by our having just generated a unique mouse model in which E46K-like, tetramer-abrogating aS mutations cause biochemical, neuropathological and motor phenotypes strikingly resembling PD. We will address 3 distinct Aims. 1. Deeply phenotype the age- and gender-related temporal development of the PD-like biochemical, neuropathological and motor syndromes of our 3K vs. 1K vs. hu WT tg mice. We will pay special attention to the emerging but unsettled evidence that clusters of membrane vesicles may be the precursors of filamentous Lewy-type bodies that are late-stage lesions. 2. Examine the role of fatty acid saturation and the key cellular desaturase, SCD, as a modulator of the normal aS tetramer:monomer equilibrium in vivo. We will use both genetic and pharmacological lowering of SCD activity to learn if this ameliorates the T:M shift of fPD mutant mice and its many downstream consequences. 3. Ask if our new hypothesis of a possible shared T:M mechanism for aS dyshomeostasis applies to three forms of fPD by a) creating the first mouse with G51D and its amplification as a ?3D? mutant; b) extending the discovery of GBA mutations in lowering the T:M ratio; and c) asking if LRRK2 mutations may also alter the aS T:M equilibrium in neurons. These distinct but thematically related Aims and the unexpected data that will flow from them should advance the basic science and therapeutics of PD & DLB.